The investigation of the bicyclo[3.1.0]hexane template as a platform for the construction of novel conformationally locked carbocyclic nucleosides continues to yield many interesting compounds. We use these modified nucleosides with the intent of: (1) determining the conformational preferences of enzymes involved in the biochemistry of nucleosides, nucleotides, and oligonucleotides; and (2) developing selected compounds as possible antitumor/antiviral drugs based on our understanding of their biochemical mechanism of action. This effort has resulted in the discovery of N-methanocarbathymidine (N-MCT), a compound active against herpes viruses 1 and 2, as well as human herpesvirus 8 associated with Kaposi sarcoma. This year N-MCT was also shown to be active against orthopoxvirus (Antiviral Res. 2007, 73, 69-77) and vaccinia virus infections (Antiviral Res. 2007, in press=doi:10.1016/j.antiviral.2007.06.005) in mice. N-MCT has been licensed and in vivo studies are continuing. Chemically synthesized N-MCT-5-triphosphate helped us demonstrate its activity as a delay chain terminator of DNA polymerases, and in particular, HIV reverse transcriptase (J. Mol. Biol. 2005, 345, 442-450). This property endowed the compound with the unique ability to overcome the excision mechanism of HIV resistance. However, N-MCT was inactive against HIV-infected cells unless the cells were transfected with herpes virus thymidine kinase (HSV-tk). The inability of N-MCT to be phosphorylated by cellular thymidine kinase (c-tk), prompted the search of new analogues that were investigated as substrates for kinases. This year we completed the synthesis of the D- and L-isomers (optical antipodes) as isomeric N-MCT analogues where both the fusion site of the cyclopropane ring and the critical 3-OH group were relocated to facilitate recognition by kinases. This approach resulted in a 2-fold increase in the formation of all three metabolites (mono-, di- and triphosphates) catalyzed by HSV-tk/thymidylate kinase and cellular diphosphate kinase). This work was recently published (J. Am. Chem. Soc. 2007, 129, 6216-6222). Unfortunately, the compound was still not phosphorylated by c-tk. Surprisingly, the L-isomer of this new analogue was the active enantiomer, and more surprisingly were two recent findings: (1) despite the high level anabolism to the 5-triphosphate, the compound was inactive against HSV-1 and HSV-2 infected cells; and (2) the compound was exquisitely active against HIV in HSV-tk- transfected cells. This means that new 5-triphosphate metabolite successfully discriminated between cellular polymerases and HIV-RT, being selective only against the latter. Future studies with the chemically synthesized 5-triphosphate of this new compound (in progress) will determine the mechanism of action. The high level of selectivity for just HIV-RT and not the cellular polymerases bodes well for this compound as a non-toxic agent since most HIV RT inhibitors have toxic side effects due to interference with cellular polymerases. This compound is relatively simple to make and efforts to make a pronucleotide to circumvent the use of HSV-tk are underway. Separately, the discovery of D-carba-T (carbocyclic thymidine) as a successfully kinased drug by ctk continues. Both the L-enantiomer and various forms of nucleotide prodrugs were shown to be active against HIV-infected cells without HSV-tk transfection. In addition, the compounds were able to block the replication of multi-drug resistant HIV-1 vectors in cultured cells. The corresponding D- and L-carbadeoxyadenosines have been recently synthesized. Our results on the use of bicyclo[3.1.0]hexane nucleosides as DNA components appeared recently in press (Nucleic Acids Res. 2007, 35, 1978-1991). The contrasting behavior and annealing properties of antipodal North- and South-containing olidodeoxynucleotides shows that small segments of DNA can be molded with specific bending or curvature. This property is being exploited with the aim of designing short DNA segments capable of binding to transcription factors. We have also completed the first synthesis of a thrombin aptamer (G-tetrad) incorporating locked North and South guanosine nucleosides. Biophysical studies (CD & NMR) are in progress